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Evaluating the Role of Serine Protease Inhibition in the Management of Tumor Micrometastases

Evaluating the Role of Serine Protease Inhibition in the Management of Tumor Micrometastases

Conservation of blood is a
priority during surgery, owing
to shortages of donor blood
and risks associated with transfusion
of blood products.[9,10] However,
blood transfusions have been linked
to a number of negative postoperative
sequelae, including poorer prognosis
after cardiac and cancer surgery.[11-
21] In this context, recognition that
allogeneic transfusion-associated
immunomodulation can increase
morbidity in allogeneically transfused
patients has become a major concern
in transfusion medicine.[9,22,23]

In cancer surgery, various studies
have documented a positive association
between transfusion and death
and relapse. In several retrospective
analyses of transfusions in colorectal
cancer surgery, long-term survival (3
to 5 years) ranged from 60% to 81%
for patients not transfused vs 37% to
63% for transfused patients (for all
studies, P < .05).[13-15] In one of
these studies, the deleterious effect of
transfusion was evident in some patients
after they received a single unit
of blood.[13] In other studies,
perioperative blood transfusion was
identified as an independent risk factor
for colorectal cancer relapse
(P = .05).[13,15] Discrepant long-term
and short-term survival rates have also
been observed in patients with esophageal
carcinoma, based on perioperative
allogeneic-blood-transfusion
status.[17,20] In most of the studies
involving esophageal resection, intraoperative
allogeneic blood transfusion
was an independent predictor of, or
prognostic covariate for, patient survival.[
9,16,18,19,21,24]

Reducing the Need for
Allogeneic Blood Transfusion

Bacterial infection, mistransfusion,
and transfusion-related acute lung injury
account for most transfusion-related
deaths, with bacterial contamination
responsible for approximately
seven deaths per million units transfused.
Although it is the third leading
cause of transfusion-related mortality,
transfusion-related acute lung injury
has been frequently underdiagnosed
and underreported, even when it occurred
in several patients receiving
transfusions from the same frequent
plasma donor.[25] Thus, transfusionrelated
acute lung injury represents a
substantial risk for patients receiving
a transfusion. Evidence from animal
models indicates that transfusion of
platelet concentrates may cause transfusion-
related acute lung injury as a
result of infusion of bioactive lipids
generated during storage.[26]

Not unexpectedly, the risk of transfusion-
related infection in cardiopulmonary
bypass patients depends on
the number of units transfused.[11]
Multiple linear and logistic regression
analyses of data from patients (n =
238) who underwent first-time coronary
artery bypass graft surgery indicated
that the amount of homologous
blood transfused was a significant and
independent predictor of postoperative
infection.[11] Infections were observed
in 3.9% of patients receiving
up to 2 units of red blood cells (RBCs)
or whole blood, 6.9% of patients receiving
3 to 5 units, and 22% of patients
receiving 6 units or more
(P = .0022). Although the risk of transmission
of infectious agents is well
recognized, the dose-response relationship
between transfusion and infection
may also be attributed to the
immunosuppressive effects of homologous
blood transfusions.[11]

A dose-dependent rate of infection
has been seen in other studies of transfused
cardiopulmonary bypass populations.[
27] Higher rates of postoperative
bacterial infections occurred in
patients transfused with packed cells
without buffy coat compared with patients
given leukocyte-depleted blood
(P = .06 overall and P = .04 for those
who received more than three transfusions).
Mortality within 60 days was
also significantly lower in patients receiving
leukocyte-depleted blood
(P = .025) compared with recipients
of packed cells, and the effect was
dose-dependent.[27]

Similarly, a recent retrospective
cohort study in 23 Canadian hospitals
found significantly lower unadjusted
in-hospital mortality rates following
the introduction of leukoreduction,
compared with the control period
(6.19% vs 7.03%, respectively;
P = .04).[28] Notably, the adjusted
odds of death following leukoreduction
were decreased (odds ratio
[OR]: 0.87; 95% confidence interval
[CI]: 0.75 to 0.99), but serious nosocomial
infections did not decrease (adjusted
OR: 0.97; 95% CI: 0.87 to 1.09)
compared with the control period.

Although leukocytes in transfused
blood can produce immunomodulatory
effects that increase the
risk of infection, the significant reduction
in mortality following leukoreduction
cannot be fully explained by
marginal changes in infection incidence
noted in the previous studies.
The precise mechanisms underlying
the link between leukoreduction and
outcome have not yet been elucidated,
but it has been postulated that transfused
leukocytes, activated during
storage, contribute to an existing inflammatory
response and exacerbate
tissue damage.[27]

Recent studies indicate that not
only are short-term mortality and morbidity
increased by blood transfusion,
but also long-term survival rates are
reduced. A review of long-term patient-
survival data (n = 1,915) from the
United States Social Security Death
Index showed that blood transfusion
during or after coronary artery bypass
graft surgery is associated with decreased
long-term survival.[12] In a
separate study, transfused cardiopulmonary
bypass patients had twice the
5-year mortality of nontransfused patients
(15% vs 7%, respectively). After
correction for comorbidities and
other factors, transfusion was still associated
with a 70% increase in mortality
(relative risk [RR]: 1.7; 95% CI:
1.4 to 2.0; P = .001). Transfusion
remained a significant predictor
(P = .04) of long-term (1- to 5-year)
mortality in multivariate analysis. Finally,
reexploration for bleeding was
identified as a strong independent
risk factor for operative mortality
(P = .005) in a separate multivariate
logistic regression analysis of data
from cardiopulmonary bypass patients
(n = 6,015).[29]

Serine Proteases in Coagulation
and Inflammation

Clearly, although the amount of
perioperative bleeding in cancer surgery
can be substantial enough in
some cases to warrant a blood transfusion,
such transfusions do have the
potential to negatively impact patient
outcomes by generating immunosuppressive
and generalized inflammatory
responses.

It is interesting that hemostasis and
inflammation share several reactants
in common and both serve as hostdefense
mechanisms.[2,30] The activation
of coagulation and inflammation
is closely linked through a network
of both humoral and cellular
components, including proteases of
the coagulation and fibrinolytic cascade.[
5] Serine proteases are essential
for virtually all inflammatory and
coagulative processes in the extracellular
or plasma phase, and they are
represented by such ubiquitous molecules
as trypsin, thrombin, plasmin,
plasminogen activator (PA), kallikrein,
and elastase.[2]

The normal physiologic response
to injury results in the generation of
procoagulants, primarily tissue factor,
which initiates thrombin generation
and clot formation, and in the generation
of plasminogen activator, which
is central to coordinated cell proliferation
and tissue remodeling.[30] Generation
of thrombin is key to activation
and release of several humoral
mediators involved in hemostasis and
inflammation.[2] A critical serine protease
of the hemostatic system, thrombin
is the final common mediator of
both the intrinsic and extrinsic coagulation
pathways, mediating the proteolytic
cleavage of fibrinogen to fibrin
and catalyzing the cross-linkage
of the fibrin clot.[31-36]

Clot formation is typically initiated
by a series of platelet-related events
that, together with blood trauma
and/or the exposure of blood to tissue
factor, promote activation of
the coagulation system.[2,33-36]
Amplification and progression of the
hemostatic system requires the presence
of an organizing surface, zymogen,
cofactor, and serine protease.[
2]

Thrombin, in addition to being a
major effector protease in the coagulation
cascade (converting fibrinogen
to fibrin), has many secondary effects.[
31,32] For example, this serine
protease triggers expression of
procoagulant activity on the platelet
surface by activating cofactors of tenase
and prothrombinase complexes, supporting
the generation of additional
thrombin.[2]

Thrombin is a direct agonist of
platelet activation and aggregation
through a protease-activated-receptor-
mediated series of events.[31,32] It
triggers platelet release of platelet agonists
such as adenosine diphosphate,
serotonin, and thromboxane, which
further amplify the platelet-activation
process, and it triggers release of
chemokines and growth factors.[2,37]
In addition, thrombin mobilizes adhesive
proteins and activates the platelet
glycoprotein (GP) IIb/IIIa receptor,
which has high affinity for fibrinogen
and von Willebrand factor.[32,38-40]

Thrombin is integral to angiogenesis
and smooth-muscle-cell proliferation,
by stimulating secretion of
growth factors such as platelet-derived
growth factor and vascular endothelial
growth factor.[31,32,41-44]
Thrombin activates inflammatory processes
and is chemotactic for monocytes
and mitogenic for lymphocytes.[
5,32,41]

Fibrinolysis and the
PlasminogenPlasmin System

Once a fibrin surface is formed, fibrinolysis
is initiated by the generation
of plasmin, a serine protease with broad
trypsin-like specificity.[1,4,45,46] Plasmin
not only is responsible for the
degradation of fibrin, fibrinogen, and
other clotting factors during clot dissolution,
but it also is capable of degrading
virtually all components of the
extracellular matrix (ECM). In addition,
it stimulates activation of other
proteases, such as MMPs and elastase,
which further degrade the extracellular
matrix.[1]

Plasmin is derived from its precursor
plasminogen (zymogen) via the
endogenous plasminogen activators
urokinase-type PA (uPA) and tissuetype
PA (tPA)[1,4,45-47] (Figure 1).
These two enzymes appear to have
different physiologic roles, with tPA
being primarily associated with clot
lysis and uPA mediating tissue-remodeling
processes.[1] Even small
amounts of plasminogen activator can
result in high local concentrations of
plasmin, through the action of uPA and
tPA. These activators are opposed by
plasminogen-activator inhibitors (PAIs),
designated PAI-1, -2, and -3, and the
activity of plasmin itself is regulated by
naturally occurring serine protease inhibitors,
such as alpha2-antiplasmin and
alpha2-macroglobulin.

Urokinase-type plasminogen activator
is secreted by a variety of both
normal and neoplastic cells as a singlechain
proenzyme (pro-uPA) with virtually
no intrinsic enzymatic activity.[
1,47] However, pro-uPA can be
activated by a variety of serine proteases,
including plasmin, kallikrein,
and trypsin-like enzymes, producing
a high-molecular-weight form of uPA
that is further degraded into enzymatically
active low-molecular-weight
uPA. Indeed, trace amounts of plasmin
are able to activate pro-uPA, thus generating
a feedback mechanism of prou
PA and plasminogen activation.[1]

The specific cellular receptor for
uPA (uPA-R) is found on a variety of
cell types and appears to play a central
role in mediating pericellular proteolytic
activity.[1,46-48] After secretion,
pro-uPA binds to uPA-R and is
activated by proteolytic cleavage to the
enzymatically active uPA form. The
interaction of uPA with uPA-R ensures
focal localization of enzyme activity
on the cell surface, and plasminogen
activation is accelerated owing to the
juxtaposition of uPA and plasminogen.
In addition to maximizing uPA and
plasminogen interactions, such binding
also impedes inactivation by naturally
occurring inhibitors.

Thus, the cell surface is the preferential
site for plasminogen activation
as uPA binds to its specific cellular
receptor. Bound uPA is more active
than unbound uPA for plasmin generation.
This arrangement is optimal
for efficient generation of pericellular
proteolytic activity.[1,47,48]

Multifunctionality of
Serine Protease Inhibitors

A single-chain polypeptide comprising
58 amino-acid residues,
aprotinin inhibits the action of numerous
serine proteases, with decreasing
affinity for trypsin, plasmin, kallikrein,
elastase, urokinase, and thrombin, respectively.
The complex pharmacodynamics
of aprotinin translates into a
dose-dependent effect on serine protease
activity. At low concentrations
(eg, about 50 kallikrein-inhibiting
units [KIU]/mL), aprotinin is a powerful
inhibitor of plasmin, but at higher
concentrations (> 200 KIU/mL) it also
possesses inhibitory activity against
kallikrein, elastase, urokinase, and
thrombin[49] (Figure 2).

Hemostatic
Properties of Aprotinin

Although the source of cardiopulmonary
bypass-induced coagulopathy
is multifactorial, platelet dysfunction
has been implicated as a primary cause
of postoperative bleeding in this setting.[
8,50,51] During extracorporeal
circulation of blood, the expression of
platelet adhesive receptors, such as
glycoprotein (GP) Ib, GP IIb, GP IIa,
and GP IIb/IIIa, is reduced. This decline
in the numbers of adhesion receptors
on the platelet surface is mediated
in part by plasmin.[52,53]
Dysregulated fibrinolysis also contributes
to the hemostatic defect that accompanies
extracorporeal circulation.[
50] During fibrinolysis, platelet
receptors bind fibrin degradation
products in place of fibrinogen, leading
to impaired platelet aggregation
and function.[51]

Aprotinin acts in a variety of interrelated
ways to reduce platelet dysfunction
by inhibiting serine proteases,
such as plasmin and kallikrein, and
preserving platelet receptors (eg, GP
Ib and others).[8,51,54] Plasmin is
directly inhibited by aprotinin, but
aprotinin also blocks contact activation
of kallikrein, which is partly responsible
for creating enzymatically
active uPA that converts plasminogen
to plasmin. These antiplasmin activities
retard the inhibitory effect of plasmin
on the expression of platelet adhesive
receptors. Furthermore, the inhibition
of plasmin by aprotinin directly
diminishes fibrinolysis, in turn
causing a reduction in fibrin/fibrinogen
degradation products, such as Ddimer,
that otherwise would impair
platelet function. Thus, the hemostatic
effect of aprotinin can be attributed to
both its inhibition of fibrinolytic activity
and its preservation of platelet
membrane-binding functions.

Clinical studies have established
that the antifibrinolytic and plateletprotective
properties of aprotinin can
decrease blood loss and transfusions
in several subsets of surgical patients.[
55-60]

Subsequent double-blind, randomized,
placebo-controlled studies confirmed
the transfusion-sparing properties
of aprotinin in primary and
reoperative cardiac surgery.[57-60]
Recent results from randomized, controlled
studies have also shown that
aprotinin decreases perioperative
bleeding and blood-transfusion requirements
in a dose-dependent fashion,
in orthopedic and transplantation
surgery as well as cancer surgery.

A study in orthopedic surgery
(n = 58), which compared "largedose"
(4 106 KIU loading dose, followed
by 1 * 106 KIU/h infusion) and
"small-dose" aprotinin (2 * 106 KIU
loading dose, followed by 5 * 105 KIU/
h infusion), showed a significant reduction
(P < .05) in postoperative
drainage in the two aprotinin groups,
compared with placebo.[61] Total
measured bleeding and total calculated
bleeding decreased significantly (both
P < .05) in the large-dose group compared
with placebo but did not achieve
statistical significance in the smalldose
group. The total number of transfused
homologous and autologous
units was also significantly decreased
(P < .05) in the large-dose aprotinin
group vs the placebo group.

In orthotopic liver transplantation
(European Multicentre Study in
Aprotinin in Liver Transplantation),
aprotinin significantly lowered intraoperative
blood loss, with a reduction
of 60% in the "high-dose" group and
44% in the "regular-dose" group compared
with placebo (P = .03 comparing
all three groups).[62,63] The
"high-dose" aprotinin regimen consisted
of a 2 * 106 KIU loading dose,
followed by 1 * 106 KIU/h infusion,
plus 1 * 106 KIU before graft
reperfusion. The "regular-dose" group
received a full Hammersmith regimen.
A comparison of these dosing schedules
showed that the total amount of
homologous and autologous RBC
transfusion requirements was 37%
lower in "high-dose" recipients and
20% lower in "regular-dose" recipients,
compared with patients in the
placebo group (P = .02, comparing all
three groups). These findings are in
line with the significant reduction
(P < .03) in transfusion requirements
with aprotinin reported in the
reoperative heart-transplantation
study.[50] Thus, aprotinin has been
shown to improve hemostasis in both
cardiac and abdominal surgery.

Studies in Cancer Patients

Importantly, significant blood- and
transfusion-sparing effects have also
been demonstrated with aprotinin in
patients undergoing resection for primary
malignant, metastatic, or benign
tumors of the liver.[64,65] In a doubleblind,
prospective, randomized study,
patients (n = 97) undergoing elective
liver resection were stratified by diagnosis
and assigned to "large-dose"
aprotinin (2 * 106 KIU loading dose,
followed by 5 * 105 KIU/h infusion,
plus a 5 * 105 KIU bolus for every 3
transfused RBC units) or placebo.
Results showed a significant overall
reduction in intraoperative blood loss
with aprotinin, compared with placebo
(mean: 1,217 vs 1,653 mL, respectively;
P = .048).[64] In stepwise logistic
regression analysis, aprotinin
treatment remained significantly correlated
with blood loss after an adjustment
for diagnosis of underlying disease,
age, preoperative hematocrit,
type of surgery, duration of clamping,
repeat surgery, and postoperative Ddimer
levels. The percentage of transfused
patients (17% vs 39%, respectively;
P = .02) and the total transfusion
requirement (30 vs 77 RBC units,
respectively; P = .015) were also significantly
lower in the aprotinin group
vs the placebo group. Given the independent
prognostic value of PAI-1 levels
in at least some tumor types,[66,67]
it is noteworthy also that the increase
in PAI levels in this study was significantly
lower with aprotinin than with
placebo (P < .0001).[64]

The overall findings of the previous
study were reproduced in a
subanalysis restricted to patients with
colorectal metastasis. In this cohort,
intraoperative blood loss (P = .037)
and transfusion requirements (P = .03)
were significantly reduced in patients
treated with aprotinin vs placebo.[65]
A similar intraoperative increase in
thrombin-antithrombin complexes
in aprotinin and placebo groups indicated
a comparable activation
of coagulation. As in the whole
study population, however, aprotinin
significantly reduced (P = .01) intraoperative
hyperfibrinolysis compared
with placebo, as measured by intergroup
comparison of D-dimer levels.

Most of the safety experience
with aprotinin has been outside
the oncology setting, in patients undergoing
cardiac surgery. Current evidence
indicates that clinically relevant
doses of aprotinin not only are generally
safe and well tolerated,[58,59,68-
71] but also are associated with lower
mortality risk in this patient population.[
71]

When considered together, ample
evidence indicates that blood transfusions
increase the risk of mortality and
relapse, and may, in fact, be an independent
risk factor for these events
following resection of some tumors.
The underlying mechanisms for these
adverse outcomes have yet to be fully
elucidated but may include transfusion-
related immunosuppression and
inflammation. Immune suppression
not only increases the risk of postoperative
infections but probably also
increases the odds of cancer relapse
and recurrence.[9] These immune-system
changes take place in a milieu of
transfusion-induced inflammation and
resulting tissue injury. Accordingly,
use of serine protease inhibitors or
other transfusion-sparing agents may
contribute to improved outcomes after
resection of intrathoracic and intra-
abdominal malignant disease.

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